With the advance of bioengineering and biotechnology, many bioactive polypeptides (proteins) and peptide medicines have been developed as therapeutic options for various diseases. Due to their low stability, however, such polypeptides or peptide medicines readily denature and thus are highly prone to renal or hepatic clearance. Accordingly, protein medicines comprising polypeptides as medicinally active ingredients suffer from the disadvantage of frequent necessary administration to patients to maintain appropriate serum levels and titers thereof. It is thus essential for the development of protein medicines that allow for them to be maintained at a proper level in the body without frequent administration.
To solve these problems, a lot of effort has been devoted to improving the serum stability of protein drugs and maintaining high drug concentration level in blood for a prolonged period of time for the maximization of the pharmaceutical efficacy of the drugs, thus improving change of protein formulations, fusion with other proteins or binding polymer have been attempted. One of the most favored methods has been focused on the fusion of immunoglobulins to proteins in recent years.
There have been many attempts made to increase the stability of protein medicines by use of immunoglobulins and their fragments, as described in U.S. Pat. No. 5,045,312 wherein human growth hormone is conjugated to bovine serum albumin or mouse immunoglobulin via a cross-linking agent. The conjugates have enhanced activity compared with unmodified growth hormone. Other various fusion proteins are also prepared as expressed in mammals after the Fc fragment of immunoglobulin is linked to interferon (Korean Patent Publication No. 10-2003-0009464), interleukin-4 receptor, interleukin-7 receptor or erythropoietin (Korean Patent No. 10-249572). PCT Patent Publication No. WO 01/03737 discloses a fusion protein in which a cytokine or a growth factor is linked through an oligopeptide linker to an Fc fragment of immunoglobulin. Also, U.S. Pat. No. 5,116,964 describes a protein which is fused to the amino or carboxy end of an immunoglobulin Fc fragment using a genetic recombination technique. U.S. Pat. No. 5,349,053 discloses a fusion protein in which IL-2 is linked to an immunoglobulin Fc fragment via a peptide linkage.
Many other Fc fusion proteins constructed using genetic recombination techniques have been disclosed, examples of which include a fusion protein of an immunoglobulin Fc fragment with interferon-beta or a derivative thereof (PCT Patent Publication No. WO 00/23472), and an immunoglobulin Fc fragment with an IL-5 receptor (U.S. Pat. No. 5,712,121). Further, an immunoglobulin Fc fragment has been used as a carrier rather than a fusion partner, as disclosed in U.S. Pat. No. 7,736,653.
Production of immunoglobulins or immunoglobulin Fc fragments has been carried out predominantly in E. coli. The American company Amgen Inc. described, in U.S. Pat. No. 6,660,843 and U.S. Pat. Publication Nos. 2004-0044188 and 2004-0053845, a human IgG1 Fc derivative having amino acid deletions at the first five amino acid residues of the hinge region, which is fused to the amino or carboxyl terminal end of a therapeutic protein or a therapeutic protein mimicked by a peptide, and the production thereof using an E. coli host. However, this fusion protein not having a signal sequence is expressed as inclusion bodies, and thus must be subjected to an additional refolding process. This protein refolding process reduces yields, and, especially in a protein present as a homodimer or a heterodimer, remarkably reduces dimer production. Also, when a protein not having a signal sequence is expressed in E. coli, a methionine residue is added to the N-terminus of the expression product due to the feature of the protein expression system of E. coli. The aforementioned expression products of Amgen Inc. have an N-terminal methionine residue, which may induce immune responses upon repeated or excessive administration to the body. Also, since these fusion molecules are expressed in a fusion protein form in E. coli by linking a gene encoding a therapeutic protein to an Fc gene they are difficult to express in E. coli, and a therapeutic protein is difficult to produce in E. coli if its expression in E. coli in a fused form results in a significant decrease or loss of activity. Further, since the fusion of two molecules creates a unnaturally-occurring abnormal amino acid sequence at the connection region between two proteins, the fusion protein could potentially be recognized as a foreign matter by the immune system, and thus induce immune responses.
As described above, the use of E. coli is advantageous in that therapeutically effective proteins can be expressed as aglycosylated forms at high yield thanks to the rapid growth rate of E. coli and the accumulated technology of fermentation and bioengineering, but disadvantageous in that the recombinant proteins have methionine as the first amino terminal residue, as opposed to native proteins, and require a complex purification process in consideration of the removal of E. coli-derived pyrogens (endotoxins) and protein refolding.
On the other hand, the use of animal cells advantageously allows for the production of fusion proteins as glycosylated proteins akin to native immunoglobulin forms, but suffers from the disadvantage of having high production cost, and being high prone to contamination with animal-derived viruses or proteins. There is therefore an increasing demand for solutions to the above-mentioned problems. Recommended is a strategy of utilizing yeasts having the advantages of both E. coli and animal cells as host cells.
Representative among the yeast used for protein production is Saccharomyces cerevisiae. In addition to being safe for the human body, the eukaryote Saccharomyces cerevisiae is easy to genetically manipulate and to culture on a large scale. Also, various expression systems for the eukaryote have been developed. When producing higher cell-derived proteins, such as human proteins, using a recombinant method, this microorganism moreover provides the proteins with the ability to be secreted outside the cells and be post-translationally modified, such as via glycosylation. In addition, the recombinant protein of the yeast undergoes folding, and disulfide bond formation, glycosylation during secretion signal-driven extracellular secretion, thus evolving to a fully bioactive form. The yeast is also economically beneficial because it does not require cell lysis and protein refolding, which are of low efficiency. What has been shown as a problem with the protein secretion system of Saccharomyces cerevisiae is, however, the great variance in secretion rate depending on the kind of human protein. Often, proteins for use as human medicines of high value are difficult to express and secrete in Saccharomyces cerevisiae (Korea Patent No. 10-0798894).